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Determinants governing the CYP74 catalysis: Conversion of allene oxide synthase into hydroperoxide lyase by site‐directed mutagenesis
Author(s) -
Toporkova Yana Y.,
Gogolev Yuri V.,
Mukhtarova Lucia S.,
Grechkin Alexander N.
Publication year - 2008
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2008.09.005
Subject(s) - mutagenesis , chemistry , allene , site directed mutagenesis , mutant , enzyme , atp synthase , lyase , biochemistry , catalysis , biosynthesis , stereochemistry , gene
Bioinformatics analyses enabled us to identify the hypothetical determinants of catalysis by CYP74 family enzymes. To examine their recognition, two mutant forms F295I and S297A of tomato allene oxide synthase LeAOS3 (CYP74C3) were prepared by site‐directed mutagenesis. Both mutations dramatically altered the enzyme catalysis. Both mutant forms possessed the activity of hydroperoxide lyase, while the allene oxide synthase activity was either not detectable (F295I) or significantly reduced (S297A) compared to the wild‐type LeAOS3. Thus, both sites 295 and 297 localized within the “I‐helix central domain” (“oxygen binding domain”) are the primary determinants of CYP74 type of catalysis.